In a recent study posted to the bioRxiv* preprint server, researchers analyzed the inhibition of stress granules (SG) formation in cells infected by the human common cold coronavirus HCOV-OC43 (HCoV-HCOV-OC43) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
Study: Nsp1 proteins of human coronaviruses HCoV-OC43 and SARS-CoV2 inhibit stress granule formation. Image Credit: dropStock/Shutterstock
This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources
Background
Host cells possess several mechanisms to detect viral infections and trigger immune defense reactions for suppressing viral replication and transmission. One such antiviral mechanism is the formation of SGs in response to viral infections.
SGs are condensates of ribonucleic acid (RNA) and proteins that cause sequestration of cellular factors and viral components essential for viral replication. However, viruses have evolved mechanisms to prevent the formation of SGs in the host. Despite the increasing interest in deciphering the role of SGs in viral replication, SG inhibition mechanisms for host protection are unclear.
About the study
In the present study, researchers investigated the ability of HCoV-HCOV-OC43 and SARS-CoV-2 to inhibit SG formation.
To assess SG dynamics during CoV infections, human embryonic kidney 158 (HEK)293A cells were infected with HCOV-OC43 and examined for the formation of SGs by immunofluorescence staining for T-cell internal antigen-related (TIAR) protein. After one to two days of infection, negligible SG formation was detected.
Thus, the team analyzed if HCOV-OC43 inhibited the formation of SGs, for which mock-infected and virus-infected cells were treated with sodium arsenite after a day of infection. Arsenite was used due to its ability to induce SG formation and eIF2α (eukaryotic translation initiation factor 2α) phosphorylation. While SG induction was observed in all the mock-infected cells, <50% of the cells infected by HCOV-OC43 demonstrated SG formation. Next, the magnitude of eIF2α phosphorylation in the HCOV-OC43-infected cells was assessed, and HCOV-OC43 efficiently inhibited eIF2α phosphorylation at 12 hours per infection (hpi) and 48 hpi.
To verify the applicability to other cell lines, the analyses were repeated in human colon cancer (HCT-8) and the bronchial epithelial (BEAS-2B) cell lines. SG formation and eIF2α phosphorylation were suppressed among the HCT-8 cells after two days of infection, reflecting slower viral replication in the cells in comparison to 293A. After one day of infection, substantially low levels of HCOV-OC43 nucleocapsid (N) protein were found in the HCT-8 cells. As observed in the 293A cells, the infected BEAS-2B cells demonstrated inhibition of SG formation and eIF2α phosphorylation within a day of infection. This indicated that the phenotypes were not restricted to entirely transformed cells and that the 293A model was appropriate for analyzing the inhibition of SG formation.
Nucleating proteins such as the Ras-GTPase-activating protein SH3-domain-binding protein 1 (G3BP1), G3BP2, T-cell internal antigen 1 (TIA-1), and (TIAR) drive condensation of SG. To assess the significance of G3BP1 in CoV replication, cells that overexpressed the enhanced green fluorescent protein (EGFP)-tagged G3BP1 were infected by HCoV-HCOV-OC43. In the study, ribopuromycylation assays and western blot analysis were also performed.
Results
In the study, SG formation was not observed in the HCoV-HCOV-OC43- and SARS-CoV-2-infected cells. Both viruses inhibited the formation of SGs induced by exogenous stress, such as treatment with sodium arsenite and eIF2α phosphorylation.
Further, in SARS-CoV-2-infected cells, a steep decline in G3BP1 levels was observed. Ectopic overexpression of N and non-structural protein 1 (Nsp1) by both viruses inhibited the formation of SGs and arsenite-induced eIF2α phosphorylation. Of note, Nsp1 of the highly pathogenic SARS-CoV-2 alone was adequate to reduce G3BP1 levels. This reduction depended on the depletion of messenger RNA (mRNA) in the cytoplasm, mediated by Nsp1 and TIAR. In the immunostaining analysis, a substantial reduction in the EGFP-expressing infected cells compared to controls was observed.
The study findings showed that both the viruses depend on the Nsp1 and N proteins for suppressing SG formation. Both HCOV-OC43 and SARS-CoV-2 dedicate >1 gene product each for SG inhibition. This indicates that viral disarming of SG responses is vital to generating a productive CoV infection.
While N proteins of SARS-CoV-2 and HCOV-OC43 act independently from eIF2α phosphorylation and downstream of translation arrest, Nsp1 proteins inhibit SG formation by suppressing eIF2α phosphorylation upstream of SG nucleation. Additionally, SARS-CoV-2 and not HCOV-OC43 infection depletes G3BP1 and disrupts the TIAR-mediated nucleocytoplasmic shuttling, contributing to SG inhibition with higher potency. Therefore, SARS-CoV-2 Nsp1-mediated host shutoff contributes, at least partially, to G3BP1 depletion and TIAR nuclear accumulation. G3BP1 overexpression significantly reduced HCOV-OC43 infection, indicative of the antiviral property of G3BP1 in CoV infections.
Conclusion
Overall, the study findings showed that HCoV-HCOV-OC43 and SARS-CoV-2 each contribute >1 gene product for the inhibition of SG formation, underpinning the importance of viral disarming of SG responses for generating productive infections. Both viruses efficiently inhibited SG formation, and the inhibition was mediated by Nsp1 and N proteins.
The findings highlight the antiviral property of G3BP1 and the presence of several mechanisms for SG suppression that are conserved between HCoV-HCOV-OC43 and SARS-CoV-2. SG formation may be an essential antiviral mechanism for host defense targeted by CoVs for efficient viral replication. Therefore, elucidating the SG inhibition mechanisms could reveal probable targets for anti-CoV therapies to inhibit viral replication by SG inhibition.
This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources
Article Revisions
- May 13 2023 - The preprint preliminary research paper that this article was based upon was accepted for publication in a peer-reviewed Scientific Journal. This article was edited accordingly to include a link to the final peer-reviewed paper, now shown in the sources section.
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